Hemolytic properties of hemocyanin from mud crab Scylla serrata.
Article Type: Report
Subject: Hemolysis and hemolysins (Observations)
Defense reaction (Physiology)
Authors: Yan, Fang
Qiao, Jie
Zhang, Yueling
Zhou, Nan
Liu, Yao
Guo, Lingling
Li, Yuanyou
Chen, Jiehui
Pub Date: 12/01/2011
Publication: Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2011 National Shellfisheries Association, Inc. ISSN: 0730-8000
Issue: Date: Dec, 2011 Source Volume: 30 Source Issue: 3
Topic: Canadian Subject Form: Defence reaction (Physiology)
Geographic: Geographic Scope: China Geographic Code: 9CHIN China
Accession Number: 278595640
Full Text: ABSTRACT Recently, infectious diseases have seriously inhibited the aquaculture of mud crab Scylla serrata in southeastern China. A better understanding of the immune molecules and defense mechanisms may be beneficial in reducing the harmful nature of these diseases. Available data show that hemocyanin (HMC) is a copper- containing respiratory protein present in the hemolymph of both mollusc and arthropod, and that it plays multiple roles in immune defense. In the current study, HMC from S. serrata (HMC-C) was isolated, and its hemolytic properties were investigated. The HMC-C shows hemolytic activities against vertebrate erythrocytes. The hemolysis displays dependency on pH, temperature, divalent cation, and HMC-C concentration. Complete hemolysis occurred at a concentration of 0.1 mg/mL, pH 5.0, and temperature of 37[degrees]C in the presence of calcium. Furthermore, all 5 subunits of HMC-C were detected in the solubilized incubation products of erythrocytes with HMC-C, and the hemolysis could be inhibited to different degrees by osmoprotectants of various molecular masses. Together, our data suggest that HMC-C mediates hemolysis by inserting all 5 subunits into the erythrocyte membrane, causing cell rupture through a colloid osmotic mechanism.

KEY WORDS: Seylla serrata, hemocyanin, hemolysis, colloid osmotic mechanism


The mud crab Scylla serrata is one of the most valuable shellfish species and the largest crab fishery in China. It has been widely cultured in brackish and seawater ponds around the coast of southeastern China for at least 100 y, and throughout the Indo-Pacific regions for more than 30 y (Shen & Lai 1994). During the past 5 y, crab farms in southeastern China have suffered from dramatic decreases in production as a result of infectious diseases caused by a variety of virus (such as reolike virus, rhabdolike virus A, picornalike virus, herpeslike virus, white spot syndrome virus), bacteria (Vibrio parahaemolyticus), and parasites (Hematodinium sp.) (Weng et al. 2007; Li et al. 2008). It is known that crustaceans, including crabs, lack a true adaptive immune response as found in vertebrates and are principally dependent on a series of nonspecific responses against foreign invaders. Therefore, the understanding of mud crab S. serrata immunology will be very helpful for the control of microbial diseases and the development of sustainable crab farming (Huang et al. 2006, Ko et al. 2007, Vaseeharan et al. 2007, Wang et al. 2007, Lin et al. 2008).

Many immune factors have been reported in crabs, including antimicrobial peptides like scygonadin from S. serrata (Huang et al. 2006, Wang et al. 2007) and crustin from Scylla paramamosain (Imjongjirak et al. 2009), antibacterial protein carcinin from Carcinus maenas (Brockton et al. 2007), prophenoloxidase (Ko et al. 2007), [alpha]2-macroglobulin (Vaseeharan et al. 2007), serine proteinases (Vaseeharan et al. 2006), antioxidant enzymes (Liu et al. 2009), and antilipopolysaccharide factor (Imjongjirak et al. 2007, Li et al. 2007, Yedery & Reddy 2009) from various crab species. Recently, hemocyanin (HMC), the respiratory protein of mollusc and arthropod, was reported as a novel important type of nonspecific innate immune defense molecule (Decker & Jaenicke 2004, Decker et al. 2007). HMC can be functionally converted into a phenoloxidaselike enzyme by different substances (Decker & Rimk 1998, Decker et al. 2001, Nagai et al. 2001). HMC and its degenerated fragments also have broad antifungal and antibacterial properties (Destoumieux-Garzon et al. 2001, Lee et al. 2003, Jiang et al. 2007). In addition, HMC can act as an antiviral agent against a variety of viruses, or delay the infection of white spot syndrome virus (WSSV) in vivo (Zhang et al. 2004a, Lei et al. 2008). Furthermore, our previous reports showed that HMC from shrimp Litopenaeus vannamei (HMC-S) can react with antihuman Ig as an antigen, bind to bacteria as an agglutinin, and bind to vertebrate erythrocytes as a hemolysin (Zhang et al. 2004b, Zhang et al. 2006, Zhang et al. 2009). HMC from mud crab S. serrata (HMC-C) can act as a lectin in specific agglutination (Yan et al. 2011). All of this suggests that HMC does, indeed, have many immune activities. However, so far, little is known about the contact hemolytic activity of HMC-C.

In this experiment we purified the HMC-C from S. serrata, and explored its hemolytic properties. Herein, the hemolytic activity of HMC-C is demonstrated and its optimal hemolysis conditions are defined. In addition, we investigated the interactions of HMC-C with chicken erythrocyte. This will increase our understanding of crab HMC and its functional role in immune defense.


Animal and Preparation of Mud Crab Sera

Mud crabs S. serrata were obtained from a local farm in Shantou, Guangdong Province, China. Haemolymph samples were taken using a fine capillary with a plunger at the axillae of the 5th swimmer leg and were allowed to clot overnight at 4[degrees]C. The sera were collected after centrifuging at 12,000g for 10 min and then stored at -20[degrees]C until analysis.

Isolation, Purification, and Identification of HMC-C

The isolation and purification of HMC-C were performed by affinity chromatography as described previously (Yan et al. 2011). Briefly, rabbit anti-HMC-S IgG (prepared by immunization in our laboratory (Zhang et al. 2006), 5 mg) was first purified via ammonium sulfate precipitation. The purified anti-HMC-S IgG was coupled covalently to CNBr-activated Sepharose 4B (0.5 g) using carbonate buffer (0.5 M NaCl, 0.1 M NaHC[O.sub.3], pH 8.3), and the matrix packed into a 2.0-mL syringe. A total of 200 [micro]L S. serrata sera were loaded into the affinity column. The unbound proteins were washed with phosphate-buffered saline (PBS; 0.01 M, pH 7.4) until absorbance at 280 nm reached baseline. The bound proteins were eluted with l0 mL glycine-HCl buffer (0.1 M, pH 2.4) and the eluates were neutralized with 1000 [micro]L Tris-HCl buffer (l M, pH 8.0). The potential HMC-C was concentrated by polyethylene glycol (PEG 20,000), and the concentration was determined using the Bradford method (Bradford 1976).

Identification of HMC-C was carried out by PAGE, SDS-PAGE, and immunoblotting as described previously (Yan et al. 2011). In brief, PAGE and SDS-PAGE were performed using 8% and 10% separating gel (pH 8.9) in Tris-glycine buffer (pH 8.3), respectively. For immunoblotting, the SDS-PAGE gel was transferred to a PVDF membrane (Millipore, Billerica, MA) with a JY-ZY3 Transblot semidry transfer apparatus (Junyi, Beijing, China) according to the manufacturer's instructions. The membrane was incubated for l h with blocking buffer (5% skim milk, 20 mM Tris, 0.15 M NaCl, pH 7.4) at room temperature, then incubated with rabbit anti-HMC-S antibodies (1:1,500 dilution) for 1 h, followed by goat anti-rabbit IgG-HRP (1:3,000 dilution) in the blocking buffer for l h. All incubations were performed at room temperature. Last, the membrane was washed and stained with substrate (3' 3-diminobenzidine, DAB) until optimum color was developed.

Preparation of Erythrocyte Suspension

Blood samples from a healthy human, mouse, rabbit, and chicken were centrifuged at 2,000 rpm for 10 min, and erythrocytes were obtained. The cells were washed 3 times with 0.01 M PBS (pH 7.4) and centrifuged at 500 rpm for 5 min. The erythrocytes were diluted to 0.5% suspension in 0.01 M PBS (pH 6.0) containing 0.15 M NaCl and 10 mM Ca[Cl.sub.2].

Determination of Hemolytic Activity

The hemolytic activity of HMC-C was determined as described previously (Hatakeyama et al. 1995). In brief, 0.1 mg/ mL HMC-C (0.9 mL) was mixed with 0.5% (v/v) erythrocyte suspension (0.3 mL) for 1 h at 37[degrees]C, unbroken cells and cell debris were removed by centrifugation at 3,500 rpm for 10 min, and hemolysis was determined by the absorbance at 540 nm in supernatants. The 0.5% (v/v) erythrocyte suspension treated with double-distilled water and PBS-[Ca.sup.2+] (0.01 M, pH 6.0) were used as 100% and 0% hemolysis controls, respectively. All samples were prepared in triplicate and data are expressed as means [+ or -] SE. The P values were determined using Student's t-test.

To investigate further the process of HMC-C-dependent hemolysis, dynamic analysis of hemolysis of chicken erythrocytes was performed. A total of 0.5% (v/v) chicken erythrocyte suspension (10 [micro]L) mixed with 0.1 mg/mL HMC-C (10 [micro]L) were plated on slides after incubation at 37[degrees]C for 15, 30, 45, and 60 min, and digital photomicrographs were captured with an Olympus BH-2 microscope.

Determination of the Optimum Parameters of Hemolytic Activity

To obtain the optimum parameters of the hemolytic activity of HMC-C, different conditions were performed.

1. pH: The following buffers containing 0.15 M NaCl and 10 mM Ca[Cl.sub.2] were used for preparation of chicken erythrocyte suspension: pH 5.0, 10 mM sodium acetate buffer; pH 6.0, 10 mM sodium phosphate buffer; pH 7.0 and 8.0, 10 mM Tris-HCl buffer.

2. Temperature: HMC-C was mixed with 0.3 mL 0.5% (v/v) chicken erythrocyte suspension and incubated at 0, 10, 20, 30, 37, 40, or 50[degrees]C for 1 h.

3. Divalent cation: HMC-C was added to chicken erythrocyte suspension supplemented with 10 mM Ca[Cl.sub.2], Mg[Cl.sub.2], Ba[Cl.sub.2, or Mn[Cl.sub.2]. Chicken erythrocyte suspension without divalent cations was used as a control.

4. HMC-C concentration: A total of 0.02, 0.04, 0.06, 0.08, or 0.1 mg/mL HMC-C was incubated with chicken erythrocyte suspension. All other procedures were carded out under the same conditions as described earlier for determination of hemolytic activity of HMC-C.

Interaction Analysis of HMC-C with Erythroeyte Membrane in Hemolysis

To investigate which subunits of HMC-C are involved in hemolysis, SDS-PAGE and immunoblotting were performed as described by Promdonkoy and Ellar (2003), with modification. Briefly, 0.5% (v/v) erythrocyte suspension (0.3 mL) was incubated with 0.1 mg/mL HMC-C (0.9 mL) at 37[degrees]C for 1 h. The erythrocyte membranes were collected by centrifugation at 3,500 rpm for 10 min and washed twice with 0.01M PBS buffer. The erythrocyte membrane pellets were solubilized in 2x protein loading buffer at room temperature before being applied to a 10% (w/v) polyacrylamide separating gel with a 3% (w/v) polyacrylamide stacking gel. The SDS-PAGE and immunoblotting analyses of the protein samples were carried out under the same conditions as described earlier.

Osmotic Protection Assay

An osmotic protection assay was performed as described by Aranda et al. (2005), with modification. Erythrocytes of chicken were suspended (0.5%, v/v) in 0.01 M PBS (pH 6.0) containing 0.15 M NaCl, 0.01 M Ca[Cl.sub.2], and one of the following osmoprotectants (0.015 M): glucose, sucrose, PEG 4000, and PEG 6000. Hemolysis was initiated by the addition of 0.9 mL 0.1 mg/mL HMC-C to 0.3 mL erythrocyte suspension with or without osmoprotectant. The hemolysis was assessed as described in the previous section. All samples were prepared in triplicate and data were expressed as means [+ or -] SE (t-test).



HMC-C was Separated by Affinity Chromatography

To investigate the hemolytic activity of HMC from mud crab S. serrata (HMC-C), HMC-C was isolated from S. serrata haemolymph using affinity chromatography. The purified HMC-C was subjected to analysis using PAGE, SDS-PAGE, and immunoblotting. Only 1 band was detected with PAGE analysis (Fig. 1A), whereas 5 bands and 3 main bands were found using SDS-PAGE and immunoblotting analysis, respectively (Fig. I B). These findings are in agreement with our previous report (Yan et al. 2011), suggesting that a good separation of mud crab S. serrata hemocyanin had been achieved.

HMC-C Showed Hemolytic Activity

HMC-C was incubated with erythrocytes of human, mouse, rabbit, or chicken at 37[degrees]C for 1 h. Hemolysis was observed with all types of erythrocytes tested, with hemolytic activities ranging from 69.7 [+ or -] 2.4% 99.5 [+ or -] 0.8% (Table 1). To investigate the process of hemolysis further, chicken erythrocytes were exposed to HMC-C (0.1 mg/mL) for 15, 30, 45, and 60 rain, and the cell lysis were processes monitored using bright-field microscopy analysis. As shown in Figure 2, fragmentation of cells was observed after 30 min of incubation. Most of the cells lysed after 60 min of incubation. In the negative control, chicken erythrocytes remained intact. These results indicate that HMC-C can function as a hemolysin.


Hemolysis Was Dependent on pH, Temperature, Divalent Cation, and HMC-C Concentration

It has been reported that hemolysins from different species require different conditions for their optimal hemolytic activities. These optimal conditions can provide some insights into the understanding of HMC-induced hemolysis and its physiological function. Here we tested the dependence of HMC hemolytic activity on pH, temperature, divalent cations, and HMC-C concentration (Fig. 3). The highest hemolysis activity (100%) was observed at pH 5.0, and decreased with pH from 5.0-8.0 (73.2 [+ or -] 2.9%; Fig. 3A). The hemolytic activity of HMC-C was weak at less than 10[degrees]C, increased with temperature from 10-37[degrees]C, and reached almost maximal activity (100%) at 37[degrees]C, and retained from 37-50[degrees]C (Fig. 3B). Species of divalent cations also affected the hemolytic activity of HMC-C. We tested the lysis of chicken erythrocyte suspension with a supplement of 10 mM Ca[Cl.sub.2], Mg[Cl.sub.2], Ba[Cl.sub.2], or Mn[Cl.sub.2]. Hemolysis was only observed in the calcium-supplemented group (93.0 [+ or -] 3.5%, n = 3); no significant lyses was detected with the other cations (Fig. 3C). Furthermore, when an excess of EDTA (a calcium chelating agent) was added to the erythrocyte suspension supplemented with 10 mM Ca[Cl.sub.2], hemolysis of HMC-C was inhibited. This result confirmed the calcium dependence of hemolysis. In addition, the hemolytic activity of HMC-C increased in a dose-dependent manner, with its concentration ranging from 0.02-0.1 mg/mL, with more than 95% activity at 0.1 mg/mL (Fig. 3D). These data indicate that HMC-C displays the characteristic features of hemolysin.


All of the Five Subunits of HMC-C Mediated Hemolysis

To determine which subunits of HMC-C are involved in hemolysis, the proteins solubilized from the chicken erythrocyte membranes treated with HMC-C were examined by SDS-PAGE and immunoblotting analysis. As shown in Figure 4, 5 proteins at approx 70, 72, 75, 76, and 80 kDa, which correspond to the 5 subunits of HMC-C as shown in Figure 1B, were found in the erythrocyte membranes treated with HMC-C, but not in the treatment with double-distilled water. Moreover, 3 proteins at approx 75, 76, and 80 kDa could react specifically with anti-HMC-S antibodies. Our results from subunit immune reaction experiments indicate that the 5 proteins are actual the 5 subunits of HMC-C, suggesting that HMC-C plays a role in erythrocyte hemolysis by all of its 5 subunits.


HMC-C-Induced Hemolysis Followed a Colloid-Osmotic Mechanism

To test whether the insertion of HMC-C causes pore formation and osmotic shock in erythrocyte cells, an osmotic protection assay was performed with chicken erythrocytes and HMC-C in the presence of several osmoprotectants. As shown in Figure 5, the presence of osmoprotectants, including glucose, sucrose, and PEG 4000 and 6000, significantly reduced the hemolysis of chicken erythrocytes. PEG 6000 showed the highest protection; cell lysis was reduced 5-fold compared with the control. These results suggest that HMC-C may form ion-permeable pores in the erythrocyte membrane, causing colloid-osmotic shock and cell rupture.



Hemolysin is a major part of the immediately effective, nonspecific, and natural defenses of most invertebrates against invading pathogens. It plays important roles as a humoral factor in the invertebrate innate immune system (Hatakeyama et al. 1995, Uechi et al. 2005). Recently, we reported that HMC-C plays an important role in innate immune reactions against invading pathogens (Yan et al. 2011). In combination with our latest finding that HMC-S can function as a hemolysin (Zhang et al. 2009), suggesting that HMC-C may have hemolytic activities. Consistently, here we also found HMC-C possessed obvious hemolytic property (Fig. 2 and Table 1). However, the hemolytic activities of HMC-C against mouse and chicken erythrocytes were even higher than that in HMC-S reported previously (Zhang et al. 2009). Together, these two studies reveal that both HMC-S and HMC-C can present an important function with hemolysis in natural defense systems against invading pathogens.

It has been reported that different hemolysins have different optimum conditions for hemolytic activity. Hatakeyama et al. (1995, 1996) showed increased hemolytic activity of CEL-III as pH increased from neutral to 10.0, and strong hemolytic activity appeared at high pH in the presence of calcium. In contrast, the hemolysis mediated by polymeric alkylpyridinium salts (polyAPS) from the marine sponge Reniera sarai is prevented by the presence of calcium (Malovrh et al. 1999). In the current study, we found that hemolysis of HMC-C is dependent on pH, temperature, divalent cation, and HMC-C concentration. The highest hemolysis activity of HMC-C occurs with a concentration of 0.1 mg/mL, at pH 5.0 and temperature 37[degrees]C in the presence of calcium (Fig. 3). Compared with the known optimal hemolytic conditions of HMC-S, HMC shared the same requirement for calcium, but the optimal pH conditions were different. The hemolysis activity of HMC-S increased with pH from 4.0-6.0, peaked at pH 6.0, then decreased from pH 6.0-10.0 (Zhang et al. 2009), whereas that of HMC-C peaked at pH 5.0 and decreased with pH from 5.0-8.0.

The immune functions of HMC have been shown to be related to its active subunit types (Decker & Rimk 1998, Lei et al. 2008, Zhang et al. 2009), even though the subunits of arthropod HMCs represent a class of similar polypeptide chains (van Holde & Miller 1995). As pointed out by Decker and Rimk (1998), the phenoloxidase activity of HMC in tarantula Eurypelma californicum is limited to the 2 subunits b and c of its 7 subunit types. Lei et al. (2008) reported that the 2 subunits of HMC from shrimp Penaeus japonicus show different functions in the anti-WSSV defense. Our previous report indicated that HMC-S may induce hemolysis as an oligomer (dimer or trimer form), and the trimer form is composed of monomers of 77 kDa (Zhang et al. 2009). To confirm the active subunit types of HMC-C involved in hemolysis, the solubilized incubation products of erythrocytes with HMC-C were investigated. Our results showed that 5 subunits of HMC-C were observed, but, unlike HMC-S, no HMC-C oligomer was found (Fig. 4). Thus, this suggests that HMC-C may mediate erythrocyte hemolysis by its 5 subunits binding tightly to erythrocyte membrane directly.

Previous studies have suggested that hemolysins can insert into the membrane and form ion-permeable pores, and the erythrocytes ruptured as a result of colloid-osmotic shock (Zhang et al. 2009). Equinatoxin II, a pore-forming toxin from the sea anemone Actinia equina, can bind to membranes and create cation-selective pores (Hong et al. 2002). Staphylococcus aureus [alpha]-hemolysin is known to bind to the lipid bilayer of target cells so that ion-permeable pores are formed in the membrane (Gouaux et al. 1994). HMC-S induces hemolysis by binding tightly to erythrocyte membrane and forming heterogeneous pores in the erythrocyte membrane (Zhang et al. 2009). Because HMC-C has similar hemolytic characteristics with HMC-S, and HMC-C could bind tightly to erythrocyte membrane in hemolysis, we hypothesized that HMC-C may have a similar mechanism as HMC-S, and may form pores in the membrane of erythrocytes to induce hemolysis. To demonstrate the inference, an osmotic protection assay was performed. The results indicated that HMC-C-induced hemolysis could be inhibited by osmoprotectants to different degrees, as in HMC-S, and the inhibiting rates of PEG 6000 against HMC-C (92%) was about 2 times more than HMC-S (48%; Fig. 5), suggesting that the size of ion-permeable pores formed by HMC-C in the erythrocyte membranes is smaller than that by HMC-S. Therefore, these data indicate that HMC-C-mediated hemolysis via colloid-osmotic mechanism is the same mechanism as HMC-S, but the process of pore formation and the size or amount of ion-permeable pores are probably heterogeneous between the 2 HMCs. These differences may be derived from their protein structure diversities.

In summary, the current study showed that HMC-C displays an important immune function in hemolytic activity and presents similar characteristics as hemolysin. Moreover, it may bind to membranes directly and create ion-permeable pores in the erythrocyte membranes to induce hemolysis. Further studies are required to characterize the diversities of pore formation mechanisms between HMC-C and HMC-S.


This work was sponsored by the National Natural Science Foundation of China (nos. 30871939 and 31072237), the Natural Science Foundation of Guangdong Province (nos. 9151064001000001 and 10251503101000002), the Science and Technology Program of Guangdong Province (nos. 2009B020309006 and 2007B020701006), the Science and Technology Program of Shantou City (no. 2011-19), and the Project for Transformation of Scientific and Technological Achievements of Guangdong Colleges and Universities (no. cgzhzd0812).


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Department of Biology and Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou, Guangdong 515063, People's Republic of China

* Corresponding author. E-mail: zhangyl@stu.edu.cn

([dagger]) Contribution is equal.

DOI: 10.2983/035.030.0338
Hemolytic activities of HMC-C against inhomogeneous erythrocytes.

                  Human (A)
Erythrocyte       ([dagger])       Human (B)          Human (AB)

Hemolytic         80.7 +2.0    70.2 [+ or -] 2.0   99.5 [+ or -] 0.8
activity (%) *

Erythrocyte           Human (O)             Mouse

Hemolytic         80.4 [+ or -] 3.8   86.4 [+ or -] 3.4
activity (%) *

Erythrocyte            Chicken             Rabbit

Hemolytic         97.9 [+ or -] 3.0   69.7 [+ or -] 2.4
activity (%) *

* Results are mean [+ or -] SE (n = 3).

([dagger]) Human blood types are indicated in parentheses.
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